The aim of this grant proposal is to elucidate at a molecular level the interaction between autoantibodies and TPO. Recombinant TPO-specific F(ab) fragments representing the repertoire of TPO antibodies in the sera of patients with autoimmune thyroid disease will be generated. From these F(ab) fragments, combinatorial libraries of IgGl, IgG4, and/or IgG2 with kappa or lambda light chains (or both) will be constructed, depending on the TPO autoantibody profile present in the individual patient's serum. TPO-specific F(ab) fragments will be analyzed for IgG gene family, affinity and specificity to TPO as well as the relative role of heavy versus light chain in TPO binding. Disease-associated antigenic domains and epitopes on TPO will also be determined. Competition studies will be performed using TPO-specific F(ab) fragments and serum TPO antibodies. The linear epitopes recognized by TPO-specific F(ab) fragments will be determined by screening with a TPO cDNA fragment library. Conformational TPO epitopes recognized by both the recombinant F(ab) fragments and TPO antibodies present in patients' sera will be determined by evaluating the interaction with a series of TPO/myeloperoxidase (MPO) chimeras to be constructed. The relationship between domains and epitopes on TPO recognized by autoantibodies, and manifestations of autoimmune thyroid disease will be evaluated.